Original research article GC-MS Analysis of Young of cobbe (L.) Raeusch. and Allophylus serratus (Roxb.) Kurz.

Chavan Reshama Bharat and Gaikwad Dattatraya Krishna Department of Botany, Shivaji University, Vidyanagar, Kolhapur, 416004, (MS) .

ABSTRACT Background: (L.) Raeusch. and Allophylus serratus (Roxb.)Kurz. () are medicinal traditionally used to cure bone fractures and other ailments in India. Objective: Present work was aimed at the identification of phytochemical compounds from young leaves of A. cobbe and A. serratus by applying gas chromatography mass spectrometry. Method: GC-MS technique was used for analysis of compounds. Results: It indicates 11 compounds from A. serratus and seven compounds from A. cobbe having mixture of terpenoids, alkanes and fatty acids. The major compounds found in A. cobbe were 1,1-diethoxy ethane (82.97%) (RT: 3.192min), phytol (7.07%) (RT: 43.690 min) and hexanoic acid (3.67%) (RT: 29.576 min) while in A. serratus 3-methyl butanol (53.16%) (RT: 3.223 min), 2-propenoic acid, 2-(dimethylamino) ethyl ester (27.08%) (RT: 13.94 min) and diisocytyl phthalate (4.49%) (RT: 52.584 min) were found in higher quantity. Phytol was detected from both species. Phytol possesses anti-radical, anti-cancer, antibacterial and anti-inflammatory properties as well as used in artificial synthesis of vitamin E and vitamin K in cosmetics and fragrance industry. Conclusion: The abundant presence of phytol in leaves of Allophylus species (7.07% in A. cobbe and 2.60% in A. serratus) suggests that these species may prove helpful in prevention of degenerative diseases and application of these herbs in cosmetics which may prove a cost effective natural alternative to current synthetic cosmetics. Presence of industrially important chemicals like phytol, 1,1-diethoxy ethane, hexanoic acid, diisocytyl phthalate, 2-propeonic acid, 2-(dimethylamino) ethyl ester underlines industrial importance of these species which needs further research on this aspect to focus in detail. Key words: GC-MS Analysis, Allophylus Cobbe, Allophylus Serratus, Young Leaves, phytol, medicinal plants.

INTRODUCTION Submission Date: 08-12-2016; Revision Date: 24-01-2017; 5 6 6 Allophylus (family Sapindaceae) having about bone fractures, rashes, and stomach ache. Accepted Date: 18-03-2017 255 species found worldwide out of which Benzylamide is isolated from leaves of DOI: 10.5530/ijper.51.3.75 1 7 nine species are found in India. A. cobbe and A. cobbe (L.) Raeusch . A. serratus is used Correspondence: A. serratus are species found in Maharashtra against bone fractures.8 A. serratus (Kurz) Dr. Chavan Reshama Bharat, (M.Sc. Ph.D. Botany) 2 9 state of India. These two species are found leaves contain β-sitostrerol and phenac- Department of Botany, in various localities of Kolhapur district of etamide, an anti-ulcer phytochemical.10 Shivaji University, Kolhapur 3 416 004, INDIA. Maharashtra State. Most of the local people Kumar et al. observed osteogenic activity Phone no: 8722793734 in Maharashtra call both the species by a of rutin isolated from stem of A. serratus E mail: [email protected] same name- Tipan (in Marathi language).4 in in vitro studies.11 Chavan and Gaikwad The fresh leaves of these species are tied reported antibacterial activity of A. serratus over the bone fractures for a faster recovery.4 and A. cobbe.12 The fresh leaves as well as dry powder of In recent years, GCMS is being a helpful leaves are also taken with small amount tool to identify the phytochemicals from of jaggery at early morning with empty the medicinal plants and various natu- stomach, for relief from joint pains.4 Leaves ral products; because it is a non-destruc- of A. cobbe are used by local people against tive, direct and fast analytical method www.ijper.org

472 Indian Journal of Pharmaceutical Education and Research | Vol 51 | Issue 3 | Jul-Sep, 2017 Chavan RB and Gaikwad DK: GC-MS analysis of young leaves of Allophylus cobbe (L.) Raeusch. and Allophylus serratus (Roxb.) Kurz. for identification of terpenoids, fatty acids and other 62,000 patterns (computer software version 1.10 beta, phytochemicals as well as a very less material Shimadzu). is required. The phytochemical information of young leaves of RESULTS AND DISCUSSION these species is scanty. Hence, present investigation was The GCMS data of A. cobbe is represented in Table 1 and aimed at characterizing chemical components of these plate 1. It indicates that it is the mixture of terpenoids, species. alkanes and fatty acids. A total of seven compounds were identified with the help of their retention indices MATERIAL AND METHODS as well as by mass spectrometric data. The sample of Collection of plant material young leaves of A. cobbe comprised of 1,1-diethoxy Young leaves of A. cobbe and A. serratus were collected ethane (82.97%), mesostilbene glycol (2.57%), 1,1-diethoxy from forest of Panhala (Kolhapur district, Maharashtra propane (0.6%), hexanoic acid (3.67%), amyl nitrite (1.13%), eicosanoic acid, ethyl ester (2%) and phytol State, India). The material was washed with water, blotted (7.07%). Major phytochemicals detected were 1,1-diethoxy to dry and kept in oven at 60° C until gaining the ethane (retained by 3.192 min.) and phytol (retained by constant weight. The completely dried material was 43.690 min. from column). taken out, finely ground with Willey’s grinding machine and these powders were kept in airtight containers until From young leaves of A. serratus, 11 compounds use. have been detected as indicated in Table 2 and chro- matogram in plate 2. It is the mixture of terpenoids Extraction of Plant Material and fatty acids. Eleven co mpounds were identified with the help of NIST library and available data. It 5 g of dry powders of young leaves of A. serratus and comprises 3methyl -1-butanol (53.16%), 2-propenoic A. cobbe were taken and extracted at 60 °C in absolute acid,2-(dimethylamino) ethyl ester (27.08%), diisocy- ethanol (Merck) up to 8 hr by using soxhlet apparatus. tyl phthalate (4.49%), palmitic acid ethyl aster (3.51%), After completion of extraction, extract was taken out, 1,1,3-triethoxy propane (3.17%), phytol (2.6%), poured in evaporating dish, kept on water bath (60ºC) n-hexadecanoic acid (1.68%), Nonanoic acid,9-(3-ex- and evaporated to dryness. Resulting residue remained anylidenecyclo propylidene)-2-hydroxy methyl ethyl at bottom of evaporating dish was dissolved in 5 ml ester (1.17%), octadecanoic acid, ethyl ester (1.15%), absolute ethanol and used for GC-MS analysis. 1,1-diethoxy-3-methyl-butane (1.01%) and cyclopentyl 2-n-hexadecylcyclopentane (0.99%). Major compounds Chromatographic analysis found were 3-methyl 1-butanol (retention time-3.223 GC-MS analysis was performed on a Shimadzu (Tokyo, min) and propenoic acid, 2-(di methylamino) ethyl ester Japan) Make QP-2010 with nonpolar 60 M RTX 5MS (retention time-13.942 min). capillary column, full scan mode, injector mode-split, Previous studies by Priya et. al. indicated presence of 22 (split ratio 1:20), quadra pole mass selective detector different compounds in ethanolic extracts of leaves of (MSD), injection temperature 220ºC, GC-MS interface A. serratus, identified by GC-MS technique, in which temperature 230ºC, the injection volume was 1μl. cycloheptasiloxane, diethyl phthalate and hexasiloxane Helium was employed as carrier gas, at a pressure of 60 were major compounds.13 Present study gives different KPa; flow rate was 1ml/min. Mass spectra were detected result from that of the study by Priya et. al. which may at 70eV. Temperature programming was set as follows: be due to the difference between type of plant material column temperature was started from 60ºC (held for (in present analysis, only young leaves have been used), 2 min) and linearly increased by 5ºC/min to 130ºC (held the temperature programming and solvent extraction for 2min); after that it was increased by 4ºC/min to procedure. Various phytochemicals have been isolated 200ºC (held for 2 min); further it was increased by 8ºC/ from different parts of Allophylus species (See appendix). min. to 250ºC (held for 10 min). Total GC running time An anti-ulcer compound phenacetamide have been was 55 min. detected from leaves of A. serratus.10 Extraction of A. serratus leaves followed by column chromatography Identification of components over silica gel revealed the presence of various com- Interpretation of mass spectra of GC-MS was con- pounds like- pinitol, quercetin, rutin, luteolin-7-O-β- ducted using the database of National Institute of D-glucopyranoside, and apigenin-4’-O-β-D-gluco-side Standard and Technology (NIST) having more than which were tested for osteogenic activity.11 In vitro

Indian Journal of Pharmaceutical Education and Research | Vol 51 | Issue 3 | Jul-Sep, 2017 473 Chavan RB and Gaikwad DK: GC-MS analysis of young leaves of Allophylus cobbe (L.) Raeusch. and Allophylus serratus (Roxb.) Kurz. osteogenic activity was studied using calvarial osteoblasts extract was tested by agar well diffusion method against obtained from Sprague Dawley rats by applying method bacteria-Staphyllococcus aureus and Bacillus subtilis.12 As of Ishizuya et al.11-14 Cultured osteoblasts at ~80% compared to cefotaxime, both aqueous and ethanolic confluence were trypsinised and treated with isolated extracts of young and mature leaves of AC and AS indi- compounds from plant extracts which indicated increase cated remarkable antibacterial activity against B. subtilis.12 in alkaline phosphatase activity due to rutin.11 Chavan A. cobbe exhibited significant nematicidal activity against and Gaikwad detected antibacterial activity of aqueous the knot nematode Meloidogynae incognita.15 and ethanolic extracts of young and mature leaves of There are some reports on phytochemical studies of A. cobbe and A. serratus.12 25 μl (1 mg/ml) each of A. cobbe. Screening of leaves and barks of A. cobbe gave

Plate 1: Chromatogram of GCMS analysis of young leaves A. Plate 2: Chromatogram of GCMS analysis of young leaves cobbe A. serratus

Table 1: GCMS analysis of leaves of A. cobbe L. Raeusch C.No. RT (min.) % Conc. MW MF Name Applications Ref. C H O 1,1- diethoxy flavoring agent in distilled 1 3.192 82.97 118.176 6 14 2 [29] ethane beverages

Phytol is used in the fragrance industry and used in cosmetics, shampoos, [28] toilet soaps, household cleaners, and detergents 2 43.690 7.07 296.53 C20H40O phytol anti-cancer [24] antimicrobial [23] anti-radical [22] anti-inflammatory [25] C H O For manufacture of artificial 3 29.576 3.67 116.16 6 12 2 hexanoic acid [30] flavors, hexyl derivatives C H O mesostilbene 4 10.367 2.57 214 14 44 2 * glycol eicosanoic acid, 5 40.789 2 340.5836 C H O * 22 44 2 ethyl ester used in heart diseases, [31] angina 6 39.864 1.13 117.15 C H ONO amyl nitrite 5 11 as an antidote in cynide [32,33] poisoning used as a reducing agent in 1,1-diethoxy 7 12.373 0.60 132 C H O the aerobic epoxidation of [34] 7 16 2 propane alkenes Abbrv-RT- Retention Time, %Conc.-percent concentration, MW—Molecular weight, MF- Molecular formula *-literature not found.

474 Indian Journal of Pharmaceutical Education and Research | Vol 51 | Issue 3 | Jul-Sep, 2017 Chavan RB and Gaikwad DK: GC-MS analysis of young leaves of Allophylus cobbe (L.) Raeusch. and Allophylus serratus (Roxb.) Kurz. negative tests for alkaloids when tested in the field, but steroids and triterpenes.17 In Vietnam, an anti-diabetic laboratory assays showed the presence of traces of compound, L-quebrachitol has been isolated from the alkaloids.16 Large scale extraction of the leaves obtained leaves of A. cobbe.18 the crude ‘alkaloid’ fraction which was found to be an In the present study, it is observed that the leaves of extremely complex mixture of non-alkaloidal material.16 both the species are rich in terpenes, alkanes, alcohols, Benzylamide has been isolated from leaves of A. cobbe.7 esters and fatty acids. The major compounds found in In a phytochemical screening of plants in Crocker A. cobbe is 1,1- diethoxy ethane and phytol while the major range, at Sabah, Malaysia, it was observed that leaves compound observed in A. serratus is 3-methyl butanol. of A. cobbe contained saponin while it lacked alkaloids, Two phytochemicals (Hexadecanoic acid, ethyl ester and

Table 2: GCMS analysis of leaves of A. serratus (Roxb.) Kurz. C.No. RT % MW MF Name Applications Ref. (min) Conc. (g/mol)

1 3.223 53.16 88.148 C5H11OH 3 methyl-1-butanol purification of nucleic Acids [35] 2-propenoic acid, in the manufacture of polymers [36] C H NO 2 13.942 27.08 143 7 13 2 2-dimethylamino) ethyl and copolymers ester additive in food packaging [37] plastic component of adhesives, [38] plasticizer of rubbers; used

3 52.584 4.49 390.56 C24H38 O4 diisocytyl phthalate as a penetration agent for fungicides; used in teethers (10.2%) and pacifiers (17.1%) used in insulation in building [39] wire

4 40.798 3.51 284.5 C18H36O2 palmitic acid ethyl aster used as lubricant, plasticizer [40]

5 12.350 3.17 176.2533 C9H20O3 1,1,3-triethoxy propane * used in the fragrance industry, [28] in cosmetics, shampoos, toilet soaps, household cleaners, and detergents

6 43.697 2.60 296.53 C20H40O phytol phytol has high antimicrobial [23] activity, high stability, low toxicity as cosmetics, hypolipodemic, [21] anxiolytic and antidepressant major part of human breast [41] milk 7 39.931 1.68 256.42 C H O n-hexadecanoic acid 16 32 2 food additive [42] anti-inflammatory [43] nonanoic acid,9-(3- * C H O hexanylidene cyclo 8 44.725 1.17 352 21 36 4 propylidene)-2-hydro xy methyl ethyl ester used as marker for excessive [44] alcohol consumption octadecanoic acid, ethyl 9 45.513 1.15 312.53 C H O 20 40 2 ester used in cosmetics, candles, [45] soaps, plastics, oil pastels, and for softening rubber food flavoring [46] 1,1-diethoxy-3-methyl- 10 8.316 1.01 160 C H O 9 20 2 butane found in beverages and gives [47] flavor compound has been detected [20] cyclopentyl 11 37.174 0.99 362 C H in leaves of highly medicinal 26 50 2-n-hexadecylcyclopentane plant Ocimum sactum

Indian Journal of Pharmaceutical Education and Research | Vol 51 | Issue 3 | Jul-Sep, 2017 475 Chavan RB and Gaikwad DK: GC-MS analysis of young leaves of Allophylus cobbe (L.) Raeusch. and Allophylus serratus (Roxb.) Kurz. nonanoic acid, 9-(3-hexanylidenecyclo propylidene)- alternative to current synthetic chemicals used in current 2-hydroxy methyl ethyl ester) from present study have cosmetic industry. Thus, the further detailed studies been previously reported from Apricot (Prunus of each compound will ascertain phytopharmaceutical armeniaca L.) detected by GC-MS technique.19 Apricot activity of A. cobbe and A. serratus. Additionally, to isolate is having therapeutic properties like antioxidant and and purify the identified bioactive compounds from antibacterial against gram positive bacteria.19 Another Allophylus may be useful to formulate the novel drugs compound from present investigation-cyclopentyl for various diseases. 2-n-hexadecylcy-clopentane has previously been iden- tified from leaves of highly medicinal plant-Ocimum SUMMARY AND CONCLUSION 20 sanctum. Phytol is found common in both the species. The major compound found in A. cobbe was industrially Phytol is an important phytochemical compound having important compound-1,1- diethoxy ethane (flavor 21 22 properties as an anxiolytic, anti-depressant, anti-radical, imparter in distilled beverages) while the major com- 23 24 25 antibacterial, anti-cancer, anti-inflammatory. Thus pound observed in A. serratus was 3-methyl butanol the abundant presence of phytol in leaves of Allophylus (used in flavors and nucleic acid purification). Presence species suggests that these species may prove helpful in of a very important compound amyl nitrite (used against prevention of degeneartive diseases. As well as, phytol is heart disorders) in A. cobbe, opens up new avenues for used in artificial synthesis of vitamin E and vitamin K26,27 investigation in cardio protective role of this species. Additionally, phytol has various applications in cosmetics An abundant presence of phytol in leaves of Allophylus and fragrance industry28 suggests the application of species suggests that these species may prove helpful in these herbs in cosmetics, which may prove a cost effective prevention of degenerative diseases. Phytol is used in

Appendix: Phytochemicals isolated from different parts of species of Allophylus Species Name of compound isolated Source Biological activity of compound Reference A. edulis Quercetin3-β-D glucoside Branches Anti-hypertensive [48] L-quebrachitol Twig Antidiabetic [49] A. cobbe Rutin Stem Osteogenic [11] L-quebrachitol Leaves Antidiabetic [18] Benzylamide Leaves Nil [48] A. laevigatus 11 acetoxy-4α-methoxy-eudesmane Fruit Nil [50] Apigenin 8-c-β rhamnopyranoside Fruit Nil [50] Carissone Fruit Nil [50] A. serratus Phenacetamide Leaves antiulcer [10] β-Sitosterol Leaves antiulcer [10] antiosteoporotic [11]

Rutin Leaves hepatoprotective [51]

radical scavenger [52] hepatoprotective [51] Quercetin Leaves anti-inflammatory, analgesic, aniti-cancer [53] tyrosinase kinase inhibitor [54] [11] hepatoprotective [55] Pinitol Leaves Anti-cancer [56] Anti-diabetic [57] [11] Luteolin-7-O-β-D-glucopyranoside Leaves Inhibits cell apoptosis [58]

[11] Apigenin-4’-O- β -D-glucoside Leaves against reflux oesophagitis gastritis [59] (Adapted from: Chavan and Gaikwad, 2016)

476 Indian Journal of Pharmaceutical Education and Research | Vol 51 | Issue 3 | Jul-Sep, 2017 Chavan RB and Gaikwad DK: GC-MS analysis of young leaves of Allophylus cobbe (L.) Raeusch. and Allophylus serratus (Roxb.) Kurz. artificial synthesis of vitamin E and vitamin K. Addi- 10. Rastogi RP, Mehrotra BN. Compendium of Medicinal Plants. New Delhi: Publication and Information Directorate; Back to cited text, 3,1995 page.23. tionally, phytol has various applications in cosmetics 11. Kumar M, Rawat P, Mishra D, Gautam AK, Pandey R, Singh D, Chattopadhyay N, and fragrance industry suggests the application of these Maurya R. Anti-osteoporotic constituents from Indian medicinal plants. herbs in cosmetics, which may prove a natural and cost Phytomedicine 2010 ;17(13):993-999. https://doi.org/10.1016/j.phymed. 2010.03.014; PMid:20554183. effective alternative to current synthetic chemicals used 12. Chavan RB, Gaikwad DK. Antibacterial activity of medicinally important two in cosmetic industry. Thus, the further detailed studies species of Allophylus- Allophylus cobbe (L.)Raeusch. and Allophylus serratus will ascertain phytopharmaceutical activity of these (Roxb.) Kurz. Journal of Pharmacognosy and Phytochemistry 2013;2(1):1-7. medicinal species. In addition, the isolation and purifica- 13. Priya ES, Selvan PS, Tamilselvan R. Phytochemical investigation of Allophylus serratus Kurz. leaves by UV and GC-MS analysis. Research tion of identified bioactive compounds from Allophylus Journal of Phytochemistry 2012; DOI:10.3923rjphyto.2012. may be useful to formulate the novel drugs for various 14. Ishizuya T, Yokose S, Hori M, Noda T, Suda T, Yoshiki S, Parathyroid diseases. In nutshell, some of the phytochemicals hormone exerts disparate effects on osteoblast differentiation depending on exposure time in rat osteoblastic cells. 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PICTORIAL ABSTRACT SUMMARY • GCMS analysis of young leaves of cobbe and A. serratus investigated • Phytol present abundantly in both species • Phytol is effective as an anticancer, antibacterial, anti-inflammatory • Major compound in A. cobbe-industrially important compound-1,1- diethoxy ethane (flavor imparter in distilled beverages) • Major compound in A. serratus-3-methyl butanol (used in flavors and nucleic acid purification).

478 Indian Journal of Pharmaceutical Education and Research | Vol 51 | Issue 3 | Jul-Sep, 2017 Chavan RB and Gaikwad DK: GC-MS analysis of young leaves of Allophylus cobbe (L.) Raeusch. and Allophylus serratus (Roxb.) Kurz.

About Authors Dr. Chavan Reshama Bharat: She has completed her Ph.D. from Dept. of Botany Shivaji University, Kolhapur. Her area of research interest is ethnomedicines, plant physilolgy, plant biochemistry, plant phytochemistry and biomedical technology

Prof. Dattatraya Krishna Gaikwad: He is the professor and Head of dept of Botany of Shivaji University, Kolhapur. He has guided several PhD and MPhil students. His area of research is plant physiology, plant biochemistry, phytomedicines, plant biotechnology. He has about 28 years of teaching experience in Botany.

Cite this article: Bharat CR, Krishna GD. GC-MS analysis of young leaves of Allophylus cobbe (L.) Raeusch. and Allophylus serratus (Roxb.) Kurz. Indian J of Pharmaceutical Education and Research. 2017;51(3):472-9.

Indian Journal of Pharmaceutical Education and Research | Vol 51 | Issue 3 | Jul-Sep, 2017 479